XBP1 governs late events in plasma cell differentiation and is not required for antigen-specific memory B cell development

Abstract

The unfolded protein response (UPR) is a stress response pathway that is driven by the increased load of unfolded proteins in the endoplasmic reticulum of highly secretory cells such as plasma cells (PCs). X box binding protein 1 (XBP1) is a transcription factor that mediates one branch of the UPR and is crucial for the development of antibody-secreting PCs. PCs represent only one class of terminally differentiated B cells, however, and little is known about the role for XBP1 in the other class: memory B cells. We have developed an XBP1(fl/fl) CD19(+/cre) conditional knockout (XBP1(CD19)) mouse to build upon our current understanding of the function of XBP1 in PC differentiation as well as to explore the role of XBP1 in memory cell development. Using this model, we show that XBP1(CD19) mice are protected from disease in an autoantibody-mediated mouse lupus model. We also identify a novel developmental stage at which B cells express the traditional PC marker CD138 (syndecan-1) but have yet to undergo XBP1-dependent functional and morphological differentiation into antibody-secreting cells. Finally, we show that memory B cells develop normally in XBP1(CD19) mice, demonstrating that XBP1-mediated functions occur independently of any memory cell lineage commitment.

Figure 1.

Female XBP1^+/+ and XBP1^CD19 mice were immunized i.p. with 100 µg MAP-DWEYS in CFA followed by booster immunizations with 100 µg MAP-DWEYS in IFA on days 14 and 28. (A and B) On day 35, serum was tested for anti-DWEYS (peptide; A) and anti-dsDNA (B) antibodies by ELISA of four serum dilutions. In both panels, each circle represents the mean ± SEM value for XBP1^+/+ (closed circles) or XBP1^CD19 (open circles) mice. (C) XBP1^+/+ and XBP1^CD19 mice from A and B were treated i.p. with 3 mg/kg LPS on days 52 and +54. Shown are representative images of IgG-specific IHC of kidney (top) and hippocampus (bottom) of mice 4–7 d after LPS treatment. XBP1^+/+ mice demonstrated anti-IgG binding to neurons in the CA1 region of the hippocampus. There was no anti-IgG binding in any of the XBP1^CD19 animals. Bars: (kidney) 200 µm; (hippocampus) 100 µm. All panels represent results from a single experiment with five mice per group. A second independent experiment reproduced these findings (not depicted).

Figure 2.

XBP1^+/+ and XBP1^CD19 mice were immunized i.p. with a single 100-µg dose of NP-KLH in alum. (A) Representative flow cytometry of cell surface CD138 (y axis) and B220 (x axis) expression in splenocytes isolated from XBP1^+/+ and XBP1^CD19 mice at days 0, 5, and 14 after immunization. Mean percentage ± SEM of B220^int CD138⁺ cells is indicated. (B) B220^int CD138⁺ cells were isolated by FACS from a subset of splenocytes at day 5 after immunization. Cytospin preparations of cells were stained with modified Wright-Giemsa stain. (C) Splenocytes were isolated from mice on day 5 after immunization and incubated with Brefeldin A BODIPY, which selectively stains ER and Golgi organelles. Shown are representative flow cytometry histograms for B220^int CD138⁺ cells (solid line), B220⁺ CD138⁻ cells (long dashed line), and unstained cells (short dashed line). Brefeldin A-BODIPY mean fluorescence intensity (MFI) ± SEM is indicated for B220^int CD138⁺ cells (no difference in B220⁺ CD138⁻ cells). All experiments were performed with individual mice in at least three independent experiments.

Figure 3.

XBP1^+/+ and XBP1^CD19 mice were immunized i.p. with a single 100-µg dose of NP-KLH in alum. At day 5 after immunization, splenocytes were isolated and sorted by FACS for B220⁺ CD138⁻ (B220) and B220^int CD138⁺ (CD138) populations. Panels show relative expression of indicated mRNA versus actin as measured by RT-PCR of cDNA prepared from indicated cell populations. In all panels, each bar represents the mean ± SEM value of individual XBP1^+/+ (open bar) and XBP1^CD19 (solid bar) mice in three independent experiments.

Figure 4.

Memory B cells are present in normal numbers in XBP1^CD19 mice. (A, left) Representative expression of IgD and NP on [PI, CD4, CD8, F4/80, GR1]^neg splenocytes of day-14 mice immunized with Ribi (adjuvant) only (left) or NP-KLH in Ribi (right). Shown are XBP1^fl/fl CD19^+/+ (XBP1^+/+) mice (top) and XBP1^fl/fl CD19^cre/+ (XBP1^CD19) littermates (bottom). The inset represents the percentage of cells within profile (mean ± SEM; n = 3). (A, right) Total number of IgD^lo NP-specific B cells from day 0 (naive BALB/c), adjuvant only (day 14 Ribi only), day 5, and day 14 NP-KLH in Ribi immunized XBP1^+/+ mice (white columns) and XBP1^CD19 (black columns) mice (mean ± SEM; n = 3). (B, left) Representative expression of B220 and CD138 at the surface of IgD^lo NP-specific B cells at days 5 (left) and 14 (right) from XBP1^+/+ mice (top) and XBP1^CD19 littermates (bottom). The inset represents the percentage of cells within profile (mean ± SEM; n = 3). (B, right) Total number of NP-specific B cells compartment B220⁺, CD79b⁺ B220^lo, or CD138⁺ at day 5 (top) or day 14 (bottom) immunized XBP1^+/+ (white columns) and XBP1^CD19 (black columns) mice (mean ± SEM; n = 3; unpaired Student's t test; P > 0.05 for all data points). (C) [PI, CD4, CD8, F4/80, GR1]^neg IgD^lo NP⁺ CD138⁺ splenocytes of day-5 and day-14 immunized mice were sorted directly ex vivo into NP-specific Ig (IgM or IgG) revealing ELISPOT assays. Positives were scored manually under a dissection microscope. Each assay was done in triplicate from three separate animals (mean ± SEM; n = 3; unpaired Student's t test; *, P ≤ 0.05).